Formulations for delivering insulin

ABSTRACT

Oral insulin formulations and processes for preparing oral insulin formulations are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of Provisional Application No.60/791,842 filed Apr. 12, 2006 and to Provisional Application No.60/857,747 filed Nov. 7, 2006, the specifications of both of which areherein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations (e.g. oralpharmaceutical formulations) containing insulin and one or more deliveryagents, e.g. (4-CNAB or sodium 4-CNAB), and methods of treating subjectssuffering from diabetes with such pharmaceutical formulations.

BACKGROUND OF THE INVENTION

There is a need for orally administered insulin that provides sufficientinsulin bioavailability, and processes for preparing such pharmaceuticalcompositions.

SUMMARY OF THE INVENTION

Methods of synthesizing pharmaceutical formulations containing insulinand a delivery agent compound have an effect on the bioavailability ofthe insulin upon administration of the pharmaceutical formulation (e.g.upon oral administration by a human). The present invention providesprocessing techniques that facilitate the delivery of insulin uponadministration with a delivery agent compound.

One embodiment of the present invention is a solid oral pharmaceuticalcomposition comprising 4-CNAB, or a pharmaceutically acceptable saltthereof, recombinant human insulin, povidone, dibasic calcium phosphate,and magnesium stearate.

Another embodiment of the present invention is a process for preparing asolid oral pharmaceutical formulation by introducing insulin or ananalog thereof to an aqueous solution that contains a delivery agentcompound, drying the solution to obtain an insulin/delivery agentpowder, optionally granulating the powder with intragranular excipients,optionally adding extragranular excipients to the powder or granules,and forming a unit dosage form from the resulting composition (e.g.,compressing the composition into tablets or filling capsules with thecomposition).

Another embodiment of the present invention is a process for preparing asolid oral insulin pharmaceutical composition by (a) preparing asolution of a delivery agent and insulin or an analog thereof; (b)freeze-drying the insulin/delivery agent solution; (c) milling theinsulin/delivery agent colyophilized powder obtained by freeze-dryingthe insulin/delivery agent solution; (d) mixing the milledco-lyophilized powder with intragranular excipients; (e) dry granulatingthe mixture formed in step (d), (f) adding extragranular excipients; and(g) forming a unit dosage form from the resulting composition (e.g.,compressing the composition into tablets or filling capsules with thecomposition). Dry granulation may be performed, for example, by rollercompaction or slugging and then milling the resulting product.

Another embodiment of the present invention is a process for preparing asolid oral insulin pharmaceutical composition by (a) preparing asolution of a delivery agent and insulin or an analog thereof; (b)performing rotary evaporation on the insulin/delivery agent solution;and (c) forming a unit dosage form from the product of step (b) (e.g.,tableting the insulin/delivery agent powder or adding theinsulin/delivery agent powder to capsules). This process may furtherinclude one or more of the steps of (d) milling the insulin/deliveryagent powder obtained by rotary evaporation; (e) mixing the milledpowder with intragranular excipients; (f) granulating the milled powderand intragranular excipients (e.g. by dry granulation), and (g) addingextragranular excipients. Dry granulation may be performed, for example,by roller compaction or slugging and milling.

Another embodiment of the present invention is a pharmaceuticalcomposition that includes (a) insulin or an insulin analog, (b) adelivery agent and (c) a gelatin, as that term is used herein (e.g.including gelatin alternatives).

Another embodiment of the present invention is a process for preparingan oral insulin pharmaceutical composition by introducing (a) a deliveryagent compound and (b) insulin or an insulin analog into gelatin or agelatin alternative. In a preferred embodiment, gelatin is optionallymilled and mixed with a delivery agent. The mixture is then granulatedusing an aqueous dispersion of insulin or an insulin analog.

Another embodiment of the present invention is a method of solubilizinginsulin or an insulin analog by introducing a delivery agent (e.g.4-CNAB or sodium 4-CNAB) to an aqueous solution and subsequently addinginsulin or an insulin analog to the delivery agent-containing solution.In one embodiment, the delivery agent is added to water, sodiumhydroxide is added to increase the pH of the solution (e.g., to increasethe pH to about 7 or 8), and then insulin is added to the pH-adjustedsolution.

Yet another embodiment is a method of treating diabetes in a subject byadministering a therapeutically effective amount of the pharmaceuticalcomposition of the present invention to the subject. The diabeticsubject can be a human suffering from Type I or Type II diabetes.Generally, the insulin preparation of the present invention does notinduce any significant incidence of antibodies. Preferably, the subjectis administered the pharmaceutical composition of the present inventionfor at least once day for at least 90 days. According to a preferredembodiment, the pharmaceutical composition is administered as adjunctivetherapy to a biguanide (such as metformin). According to anotherpreferred embodiment, the pharmaceutical composition is administered asadjunctive therapy to a biguanide (such as metformin), acarbose, aglitazone (e.g., pioglitazone), or a combination thereof.

One embodiment is a method of treating diabetes (e.g. type II diabetes)in a subject who has not sufficiently responded to metforminmonotherapy, by administering a therapeutically effective amount of thepharmaceutical composition of the present invention to the subject.According to a preferred embodiment, the subject continues treatmentwith metformin while also being treated with the pharmaceuticalcomposition of the present invention. According to another preferredembodiment, the pharmaceutical composition is administered as adjunctivetherapy to a biguanide (such as metformin), acarbose, a glitazone (e.g.,pioglitazone), or a combination thereof.

Yet another embodiment is a method of treating diabetes (e.g., Type I orType II diabetes) in a human having a hemoglobin A1c value of at leastabout 8.0% by administering a therapeutically effective amount of thepharmaceutical composition of the present invention to the human.According to one embodiment, the human has a hemoglobin A1c valueranging from about 8.0 to about 9.3%. Preferably, the human isadministered the pharmaceutical composition of the present invention forat least once day for at least 90 days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the solubility of recombinant human zinc insulin atdifferent pH and time-points.

FIG. 2 sets forth a solubility curve for insulin and 4-CNAB.

FIGS. 3 and 5 set forth insulin dissolution profiles in deionized water.

FIGS. 5-12 set forth results of insulin dosage forms administered tomonkeys as described in Example 7.

FIGS. 13 and 14 set forth the results of insulin dosage formsadministered to humans as described in Example 23.

FIG. 15 sets forth the results of insulin dosage forms administered tohumans as described in Example 24.

FIG. 16 sets forth the results of insulin dosage forms administered tohumans as described in Example 25.

FIGS. 17-24 sets forth the results of insulin dosage forms administeredto humans as described in Example 26.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein and in the appended claims, the singular forms “a” “an”and “the” also includes plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a molecule”includes one or more of such molecules, “a reagent” includes one or moreof such different reagents, reference to “an antibody” includes one ormore of such different antibodies, and reference to “the method”includes reference to equivalent steps and methods known to those ofordinary skill in the art that could be modified or substituted for themethods described herein.

The term “hydrate” as used herein includes, but is not limited to, (i) asubstance containing water combined in molecular form and (ii) acrystalline substance containing one or more molecules of water ofcrystallization or a crystalline material containing free water.

The term “solvate” as used herein includes, but is not limited to, amolecular or ionic complex of molecules or ions of a solvent withmolecules or ions of the delivery agent compound or salt thereof, orhydrate or solvate thereof.

The term “delivery agent” refers to any of the delivery agent compoundsdisclosed or incorporated by reference herein.

The terms “alkyl”, “alkoxy”, “alkylene”, “alkenylene”, “alkyl(arylene)”,and “aryl(alkylene)” include, but are not limited to, linear andbranched alkyl, alkoxy, alkylene, alkenylene, alkyl(arylene), andaryl(alkylene) groups, respectively.

Unless otherwise specified, the term “substituted” as used hereinincludes, but is not limited to, substitution with any one or anycombination of the following substituents: halogens, hydroxide, C₁-C₄alkyl, and C₁-C₄ alkoxy.

The term “4-MOAC” refers to8-(N-2-hydroxy-4-methoxybenzoyl)-aminocaprylic acid and pharmaceuticallyacceptable salts thereof. Unless otherwise noted, the term “4-MOAC”refers to all forms of 4-MOAC, including, but not limited to, amorphousand crystalline forms of 4-MOAC.

The term “NAC” as used herein refers to N-(8-[2-hydroxybenzoyl]-amino)caprylic acid and pharmaceutically acceptable salts thereof, includingits monosodium salt. Unless otherwise noted, the term “NAC” refers toall forms of NAC, including, but not limited to, all amorphous andcrystalline forms of NAC. The term “SNAC” as used herein refers to themonosodium salt of NAC, including, but not limited to, all amorphous andcrystalline forms of SNAC (such as those described in InternationalPublication No. WO 2005/107462, which is hereby incorporated byreference), unless otherwise indicated.

The term “NAD” as used herein refers to N-(10-[2-hydroxybenzoyl]-amino)decanoic acid and pharmaceutically acceptable salts thereof, including,but not limited to, its monosodium salt. Unless otherwise noted, theterm “NAD” refers to all forms of NAD, including, but not limited to,all amorphous and crystalline forms of NAD. The term “SNAD” as usedherein refers to the monosodium salt of NAD, including, but not limitedto, all amorphous and crystalline forms of SNAC.

The term “5-CNAC” refers toN-(8-[2-hydroxy-5-chlorobenzoyl]-amino)octanoic acid (also known as8-(N-2-hydroxy-5-chlorobenzoyl)aminocaprylic acid)) and pharmaceuticallyacceptable salts thereof, including, but not limited to, its monosodiumsalt and disodium salt. Unless otherwise noted, the term “5-CNAC” refersto all forms of 5-CNAC, including, but not limited to, all amorphous andcrystalline forms of it (including those described in InternationalPublication No. WO 00/59863, PCT/US2006/036455, filed Sep. 18, 2006, andU.S. Provisional Application No. 60/718,829, filed Sep. 19, 2005, all ofwhich are hereby incorporated by reference).

The term “4-CNAB” refers to4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate (also known as4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid) and pharmaceuticallyacceptable salts thereof, including, but not limited to, its monosodiumsalt. Unless otherwise noted, the term “4-CNAB” refers to all forms of4-CNAB, including, but not limited to, all amorphous and crystallineforms of 4-CNAB. The term “mono-sodium 4-CNAB” refers to monosodium4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate, including, but notlimited to, anhydrous, monohydrate, and isopropanol solvates thereof andamorphous and crystalline forms thereof (including those described inInternational Publication Nos. WO 02/02509 and WO 03/057650, both ofwhich is hereby incorporated by reference), unless otherwise indicated.

The term “HPOD” refers to 8-(2-hydroxyphenoxy)octyldiethanolamine andpharmaceutically acceptable salts thereof, including, but not limitedto, its meslyate salt. Unless otherwise noted, the term “HPOD” refers toall forms of HPOD, including, but not limited to, all amorphous andcrystalline forms of HPOD and includes anhydrous, monohydrate, andisopropanol solvates of HPOD, including those described in InternationalPublication No. WO 2005/115406, which is hereby incorporated byreference).

The term “insulin analog” as used herein, refers to analogs of naturallyoccurring insulins, including human insulin or animal insulins, whichdiffer by substitution of at least one naturally occurring amino acidresidue with other amino acid residues and/or addition/removal of atleast one amino acid residue from the corresponding, otherwiseidentical, naturally occurring insulin. Insulin analogs have aphysiological effect similar to that of naturally occurring orrecombinant human insulin. The added and/or replaced amino acid residuescan also be those which do not occur naturally. Insulin analogs includethose analogs disclosed in U.S. Pat. Nos. 6,960,561, 6,906,028,6,852,694, 6,777,207, 6,630,348, 6,551,992, 6,534,288, 6,531,448,RE37,971, 6,465,426, 6,444,641, 6,335,316, 6,268,335, 6,051,551,6,034,054, 5,970,973, 5,952,297, 5,922,675, 5,888,477, 5,873,358,5,747,642, 5,693,609, 5,650,486, 5,646,242, 5,547,929, 5,504,188,5,474,978, 5,461,031, 5,135,866, 4,421,685, all of which are herebyincorporated by reference.

The term “subject” as used herein includes a mammal, preferably a human.It may also mean other animals, including other mammals, but especiallybirds, poultry or other avian forms.

The phrase “pharmaceutically acceptable” refers to components orcompositions that are physiologically tolerable.

The term “treating” or “treatment” of a state, disorder or conditionincludes:

(1) preventing or delaying the appearance of clinical symptoms of thestate, disorder or condition developing in a subject that may beafflicted with or predisposed to the state, disorder or condition butdoes not yet experience or display clinical or subclinical symptoms ofthe state, disorder or condition;

(2) inhibiting the state, disorder or condition, i.e., arresting orreducing the development of the disease or at least one clinical orsubclinical symptom thereof; or

(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or subclinicalsymptoms.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the subject or to the physician.

The term “mean”, when preceding a pharmacokinetic value (e.g., meanpeak) represents the arithmetic mean value of the pharmacokinetic valueunless otherwise specified.

The term “Serum Concentration” or “Serum Concentration Curve” is thegraphic representation of the amount of drug in an animal's (includinghumans) plasma at particular points in time.

The term “Area Under the Curve” or “Area Under the Concentration Curve”or “AUC” means the area present beneath the line of the graphicalrepresentation of plasma concentrations versus time in subject(s).Unless otherwise specified, AUC refers to the AUC obtained based onbaseline adjusted concentrations, i.e., concentrations obtained aftersubtracting the individual baseline from each individual time point(C_(t)−C₀).

The term “Cmax” refers to the maximum observed concentration takendirectly from the plasma concentration-time course profile. Unlessotherwise specified, Cmax refers to Cmax obtained based on baselineadjusted concentrations, i.e., concentrations obtained after subtractingthe individual baseline from each individual time point (C_(t)−C₀).

Delivery Agent Compounds

In one embodiment of the present invention, the delivery agent compoundhas the following structure, or a pharmaceutically acceptable saltthereof:

wherein

-   -   Ar is phenyl or naphthyl;    -   Ar is optionally substituted with one or more of —OH, halogen,        C₁-C₄ alkyl, C₁-C₄ alkenyl, C₁-C₄ alkoxy or C₁-C₄ haloalkoxy;    -   R⁷ is C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl, phenyl, naphthyl, (C₁-C₁₀        alkyl)phenyl, (C₁-C₁₀ alkenyl)phenyl, (C₁-C₁₀ alkyl)naphthyl,        (C₁-C₁₀ alkenyl)naphthyl, phenyl(C₁-C₁₀ alkyl), phenyl(C₁-C₁₀        alkenyl), naphthyl(C₁-C₁₀ alkyl), or naphthyl(C₁-C₁₀ alkenyl);    -   R⁸ is hydrogen, C₁ to C₄ alkyl, C₂ to C₄ alkenyl, C₁ to C₄        alkoxy, or C₁-C₄ haloalkoxy;    -   R⁷ is optionally substituted with C₁ to C₄ alkyl, C₂ to C₄        alkenyl, C₁ to C₄ alkoxy, C₁-C₄ haloalkoxy, —OH, —SH, —CO₂R₉, or        any combination thereof;    -   R⁹ is hydrogen, C₁ to C₄ alkyl, or C₂ to C₄ alkenyl; and    -   R⁷ is optionally interrupted by oxygen, nitrogen, sulfur or any        combination thereof.

In one embodiment, the delivery agent compounds are not substituted withan amino group in the position alpha to the acid group.

Suitable delivery agent compounds include, but are not limited to,N-(8-[2-hydroxybenzoyl]-amino)caprylic acid and salts thereof, e.g., asodium salt of N-(8-[2-hydroxybenzoyl]-amino)caprylic acid, such as amono- or di-sodium salt, N-(8-[2-hydroxybenzoyl]-amino) decanoic acidand pharmaceutically acceptable salts thereof, including its monosodiumsalt, 4-[(4-chloro-2-hydroxy-benzoyl)amino]butanoic acid (also known as4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoate) and pharmaceuticallyacceptable salts thereof, including its sodium salt (e.g., monosodiumsalt), N-(8-[2-hydroxy-5-chlorobenzoyl]-amino)octanoic acid (also knownas 8-(N-2-hydroxy-5-chlorobenzoyl)aminocaprylic acid)) andpharmaceutically acceptable salts thereof, including its monosodiumsalt, and 8-(N-2-hydroxy-4-methoxybenzoyl)-aminocaprylic acid andpharmaceutically acceptable salts thereof, including its monosodiumsalt.

According to one embodiment, R⁷ in Formula A is selected from C₈-C₂₀alkyl, C₅-C₂₀ alkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl)phenyl, (C₁-C₁₀alkenyl)phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₁-C₁₀ alkenyl)naphthyl,phenyl(C₁-C₁₀ alkyl), phenyl(C₁-C₁₀ alkenyl), naphthyl(C₁-C₁₀ alkyl),and naphthyl(C₁-C₁₀ alkenyl).

According to another embodiment, R⁷ in Formula A is selected from C₈-C₂₀alkyl, and C₈-C₂₀ alkenyl.

In another embodiment of the present invention, the delivery agentcompound has the following structure, or a pharmaceutically acceptablesalt thereof:

wherein

-   -   R¹, R², R³, and R⁴ are independently H, —OH, halogen, C₁-C₄        alkyl, C₂-C₄ alkenyl, C₁-C₄ alkoxy, —C(O)R⁸, —NO₂, —NR⁹R¹⁰, or        —N⁺R⁹R¹⁰R¹¹(R¹²)⁻;    -   R⁵ is H, —OH, —NO₂, halogen, —CF₃, —NR¹⁴R¹⁵, —N⁺R¹⁴R¹⁵R¹⁶(R¹³)⁻,        amide, C₁-C₁₂ alkoxy, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, carbamate,        carbonate, urea, or —C(O)R¹⁸;    -   R⁵ is optionally substituted with halogen, —OH, —SH, or —COOH;    -   R⁵ is optionally interrupted by O, N, S, or —C(O)—;    -   R⁶ is a C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, or arylene;    -   R⁶ is optionally substituted with a C₁-C₄ alkyl, C₂-C₄ alkenyl,        C₁-C₄ alkoxy, —OH, —SH, halogen, —NH₂, or —CO₂R₈;    -   R⁶ is optionally interrupted by O or N;    -   R⁷ is a bond or arylene;    -   R⁷ is optionally substituted with —OH, halogen, —C(O)CH₃,        —NR¹⁰R¹¹, or —N⁺R¹⁰R¹¹R¹²(R¹³)⁻;    -   each occurrence of R⁸ is independently H, C₁-C₄ alkyl, C₂-C₄        alkenyl, or —NH₂;    -   R⁹, R¹⁰, R¹¹, and R¹² independently H or C₁-C₁₀ alkyl;    -   R¹³ is a halide, hydroxide, sulfate, tetrafluoroborate, or        phosphate;    -   R¹⁴, R¹⁵ and R¹⁶ are independently H, C₁-C₁₀ alkyl, C₁-C₁₀ alkyl        substituted with —COOH, C₂-C₁₂ alkenyl, C₂-C₁₂ alkenyl        substituted with —COOH, or —C(O)R¹⁷;    -   R¹⁷ is —OH, C₁-C₁₀ alkyl, or C₂-C₁₂ alkenyl; and    -   R¹⁸ is H, C₁-C₆ alkyl, —OH, —NR₁₄R₁₅, or N⁺R¹⁴R¹⁵R¹⁶(R¹³)⁻.

In one particular embodiment, when R¹, R², R³, R⁴, and R⁵ are H, and R⁷is a bond then R⁶ is not a C₁-C₆, C₉ or C₁₀ alkyl.

In another embodiment, when R¹, R², R³, and R⁴ are H, R⁵ is —OH, and R⁷is a bond then R⁶ is not a C₁-C₃ alkyl.

In yet another embodiment, when at least one of R¹, R², R³, and R⁴ isnot H, R⁵ is —OH, and R⁷ is a bond, then R⁶ is not a C₁-C₄ alkyl.

In yet another embodiment, when R¹, R², and R³ are H, R⁴ is —OCH₃, R⁵ is—C(O)CH₃, and R⁶ is a bond then R⁷ is not a C₃ alkyl.

In yet another embodiment, when R¹, R², R⁴, and R⁵ are H, R³ is —OH, andR⁷ is a bond then R⁶ is not a methyl.

In yet another embodiment, R⁶ of Formula B is a C₈-C₁₂ alkylene, C₈-C₁₂alkenylene, or arylene.

In yet another embodiment of the present invention, the delivery agentcompound has the following structure or a pharmaceutically acceptablesalt thereof:

wherein

R¹, R², R³, R⁴ and R⁵ are independently H, —CN, —OH, —OCH₃, or halogen,at least one of R¹, R², R³, R⁴ and R⁵ being —CN; and

R⁶ is a C₁-C₁₂ linear or branched alkylene, a C₁-C₁₂ linear or branchedalkenylene, a C₁-C₁₂ linear or branched arylene, an alkyl(arylene) or anaryl(alkylene).

According to one embodiment, when R¹ is —CN, R⁴ is H or —CN, and R², R³,and R⁵ are H, then R⁶ is not methylene ((CH₂)₁).

In another embodiment, R⁶ of Formula C is a C₈-C₁₂ linear or branchedalkylene, a C₈-C₁₂ linear or branched alkenylene, an arylene, analkyl(arylene) or an aryl(alkylene).

In yet another embodiment, R⁶ of Formula C is a C₈-C₁₂ linear orbranched alkylene, a C₈-C₁₂ linear or branched alkenylene.

Other suitable delivery agent compounds are disclosed in U.S. Pat. No.6,627,228, which is hereby incorporated by reference.

The delivery agent compound can also be a polymeric delivery agentcomprising a polymer conjugated to a modified amino acid or derivativethereof via a linkage group selected from the group consisting of—NHC(O)NH—, —C(O)NH—, —NHC(O)—, —OOC—, —COO—, —NHC(O)O—, —OC(O)NH—,—CH₂NH—, —NHCH₂—, —CH₂NHC(O)O—, —OC(O)NHCH₂—, —CH₂NHCOCH₂O—,—OCH₂C(O)NHCH₂—, —NHC(O)CH₂O—, —OCH₂C(O)NH—, —NH—, —O—, andcarbon-carbon bond. In one embodiment, the polymeric delivery agent isnot a polypeptide or polyamino acid. In another embodiment, the modifiedamino acid has the structure of formula A, B, or C. In one embodiment,the polymeric delivery agent includes a modified amino acid having thestructure:

-   -   which is conjugated via a —COO group to a polymer having        monomers derived from polyethylene glycol.

In one embodiment, the polymeric delivery agent is a modified amino acidhaving the structure of Formula D conjugated via a —COO group to apolymer having the structure:

—CH₂CH₂O(CH₂CH₂O)_(x)CH₂CH₂O—Y,

wherein

x is from 1-14; and

Y is H or CH₃.

According to another embodiment, the polymeric delivery agent iscompound having the structure of Formula D conjugated via a —COO groupto a polymer having the structure:

—CH₂CH₂O(CH₂CH₂O)_(x)CH₂CH₂O—Y,

wherein

x is 1-5; and

Y is CH₃ or H.

For example, the polymeric delivery agent can be8-(2-hydroxybenzoylamino)-octanoic acid2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-ethoxy)ethoxy]ethoxy}ethylester.

Other suitable delivery agent compounds include compounds of the formulabelow and pharmaceutically acceptable salts thereof:

R₁ is —(CH₂)_(m)—R₈, wherein m is 0 or 1;

R₂-R₆ are independently selected from hydrogen, hydroxyl, halogen, C₁-C₄alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C₁-C₄ alkoxy, and cyano;

R₇ is selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl;

R₈ is selected from cyclopentyl, cyclohexyl and phenyl, wherein when R₈is phenyl, m is 1; and

R₈ is optionally substituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, halogen orhydroxyl, or a combination thereof.

Other delivery agent compounds of the present invention include those ofthe formula:

and pharmaceutically acceptable salts thereof, wherein:

R₁ is a C₁-C₆ alkyl, or C₂-C₆ alkenyl,

R₂-R₆ are independently selected from hydrogen, hydroxyl, halogen, C₁-C₄alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C₁-C₄ alkoxy, and cyano, and

R₇ is selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl.

Other suitable delivery agent compounds include those of the formula:

and pharmaceutically acceptable salts thereof, wherein

n is 1 to 9, and

R¹ to R⁵ are independently hydrogen, C₁ to C₆ alkyl, C₁ to C₆ alkoxy, C₂to C₆ alkenyl, halogen, hydroxyl, —NH—C(O)—CH₃, or —O—C₆H₅.

In one embodiment, R¹ to R⁵ of Formula G are independently hydrogen, C₁to C₄ alkyl, C₁ to C₄ alkoxy, C₂ to C₄ alkenyl, halogen, or hydroxyl.

Other suitable delivery agent compounds include those of the formula:

and pharmaceutically acceptable salts thereof, wherein

R¹ to R⁴ are independently hydrogen, C₁ to C₄ alkyl, C₂ to C₄ alkenyl,halogen, C₁ to C₄ alkoxy, or hydroxyl.

Other delivery agent compounds of the present invention include those ofthe formula:

and pharmaceutically acceptable salts thereof, wherein

one of R¹ to R⁵ is —(CH₂)_(n)—COOH where n is 0-6; and

the remaining four members of R¹ to R⁵ are independently hydrogen, C₁ toC₄ alkyl, C₂ to C₄ alkenyl, halogen, C₁ to C₄ alkoxy, or hydroxyl; and

R₆-R₁₀ are independently hydrogen, C₁ to C₄ alkyl, C₂ to C₄ alkenyl,halogen, C₁ to C₄ alkoxy, or hydroxyl.

Other delivery agents of the present invention include compoundsrepresented by the formula:

and pharmaceutically acceptable salts thereof, wherein

n is 1 to 9; and R₁ to R₉ are independently hydrogen, C₁ to C₄ alkyl, C₂to C₄ alkenyl, halogen, C₁ to C₄ alkoxy, or hydroxyl.

Other suitable delivery agent compounds include those of the formula:

and pharmaceutically acceptable salts thereof, wherein

R¹-R⁵ are independently hydrogen, C₁ to C₄ alkyl, C₂ to C₄ alkenyl,halogen, C₁ to C₄ alkoxy, hydroxyl, or —O—(CH₂)_(n)—COOH (where n is 1to 12);

at least one of R¹ to R⁵ is —O—(CH₂)_(n)—COOH where n is 1-12; and

R⁶-R¹⁰ are independently hydrogen, C₁ to C₄ alkyl, C₂ to C₄ alkenyl,halogen, C₁ to C₄ alkoxy, or hydroxyl.

Suitable delivery agents are described in International. PublicationNos. WO 2005/117854 and WO 2005/112633, both of which were filed May 16,2005 and their priority documents, U.S. Provisional Application Nos.60/576,088, filed Jun. 1, 2004, U.S. Provisional Application No.60/576,397, filed Jun. 1, 2004, U.S. Provisional Application No.60/576,105, filed Jun. 1, 2004, U.S. Provisional Application No.60/571,090, filed May 14, 2004, U.S. Provisional Application No.60/571,092, filed May 14, 2004, U.S. Provisional Application No.60/571,195, filed May 14, 2004, U.S. Provisional Application No.60/571,194, filed May 14, 2004, U.S. Provisional Application No.60/571,093, filed May 14, 2004, U.S. Provisional Application No.60/571,055, filed May 14, 2004, U.S. Provisional Application No.60/571,151, filed May 14, 2004, U.S. Provisional Application No.60/571,315, filed May 14, 2004, U.S. Provisional Application No.60/571,144, filed May 14, 2004, and U.S. Provisional Application60/571,089, filed May 14, 2004, all of which are hereby incorporated byreference in their entirety.

Other suitable delivery agents include those having the followingstructure and pharmaceutically acceptable salts thereof:

wherein

(a) R¹, R², R³, and R⁴ are independently H, —OH, halogen, C₁-C₄ alkyl,C₁-C₄ alkenyl, C₁-C₄ alkoxy, —C(O)R⁸, —NO₂, —NR⁹R¹⁰, or —N⁺R⁹R¹⁰R¹¹(Y⁻);

-   -   R⁸ is hydrogen, —OH, C₁-C₆ alkyl, C₁-C₄ alkyl substituted with        halogen or —OH, C₂-C₄ alkenyl unsubstituted or substituted with        halogen or —OH, or —NR¹⁴R¹⁵;    -   R⁹, R¹⁰, and R¹¹ are independently hydrogen, oxygen, C₁-C₄ alkyl        unsubstituted or substituted with halogen or —OH, C₂-C₄ alkenyl        unsubstituted or substituted with halogen or —OH;    -   Y is halide, hydroxide, sulfate, nitrate, phosphate, alkoxy,        perchlorate, tetrafluoroborate, carboxylate, mesylate, fumerate,        malonate, succinate, tartrate, acetate, gluconate, maleate;    -   R⁵ is H, —OH, —NO₂, halogen, CF₃, —NR¹⁴R¹⁵, —N⁺R¹⁴R¹⁵R¹⁶(Y⁻),        amide, C₁-C₁₂ alkoxy, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, carbamate,        carbonate, urea, or —C(O)R²²; R⁵ is optionally substituted with        halogen, —OH, —SH, or —COOH; R⁵ is optionally interrupted by O,        N, S, or —C(O)—;    -   R¹⁴, R¹⁵, and R¹⁶ are independently H or C₁-C₁₀ alkyl;    -   R²² is H, C₁-C₆ alkyl, —OH, —NR¹⁴R¹⁵;    -   R⁶ is substituted or unsubstituted C₁-C₁₆ alkylene, C₂-C₁₆        alkenylene, C₂-C₁₆ alkynylene, C₅-C₁₆ arylene, (C₁-C₁₆ alkyl)        arylene or aryl(C₁-C₁₆ alkylene); R⁶ is optionally substituted        with C₁-C₇ alkyl or C₁-C₇ cycloalkyl;    -   R⁷ is —NR¹⁸R¹⁹ or —N⁺R¹⁸R¹⁹R²⁰Y⁻;    -   R¹⁸ and R¹⁹ are independently hydrogen, oxygen, hydroxy,        substituted or unsubstituted C₁-C₁₆ alkyl, substituted or        unsubstituted C₂-C₁₆ alkenyl, substituted or unsubstituted        C₂-C₁₆ alkynyl, substituted or unsubstituted aryl, substituted        or unsubstituted alkylcarbonyl (e.g. substituted or        unsubstituted (C₁₋₆ alkyl)carbonyl), substituted or        unsubstituted arylcarbonyl, substituted or unsubstituted        alkanesulfinyl (e.g. substituted or unsubstituted (C₁₋₆        alkane)sulfinyl), substituted or unsubstituted arylsulfinyl,        substituted or unsubstituted alkanesulfonyl (e.g. substituted or        unsubstituted (C₁₋₆ alkane)sulfonyl), substituted or        unsubstituted arylsulfonyl, substituted or unsubstituted        alkoxycarbonyl (e.g. substituted or unsubstituted (C₁₋₆        alkoxy)carbonyl), or substituted or unsubstituted        aryloxycarbonyl, or substituted or unsubstituted C₅-C₇        heterocyclic ring (i.e., 5, 6, or 7-membered heterocyclic ring),        wherein the substitutions may be halogen or —OH; and    -   R²⁰ is independently hydrogen, substituted or unsubstituted        C₁-C₁₆ alkyl, substituted or unsubstituted C₂-C₁₆ alkenyl,        substituted or unsubstituted C₂-C₁₆ alkynyl, substituted or        unsubstituted aryl, substituted or unsubstituted alkylcarbonyl        (e.g. substituted or unsubstituted (C₁₋₆ alkyl)carbonyl),        substituted or unsubstituted arylcarbonyl, substituted or        unsubstituted alkanesulfinyl (e.g. substituted or unsubstituted        (C₁₋₆ alkane)sulfinyl), substituted or unsubstituted        arylsulfinyl, substituted or unsubstituted alkanesulfonyl (e.g.        substituted or unsubstituted (C₁₋₆ alkane)sulfonyl), substituted        or unsubstituted arylsulfonyl, substituted or unsubstituted        alkoxycarbonyl (e.g. substituted or unsubstituted (C₁₋₆        alkoxy)carbonyl), or substituted or unsubstituted        aryloxycarbonyl; or

(b) R¹-R¹⁶ and R²⁰ are as defined above; and

-   -   R¹⁸ and R¹⁹ combine to form a 5, 6, or 7-membered heterocyclic        ring optionally interrupted with an oxo group and unsubstituted        or substituted with C₁-C₆ alkyl, C₁-C₆ alkoxy, aryl, aryloxy, or        carbocyclic ring.

According to one embodiment, R⁷ is morpholino, morpholinium salt, ordiethanolamino.

According to another embodiment, R⁶ is a C₁-C₁₆ alkylene and R⁷ ismorpholino or a morpholinium salt. Preferably, R⁶ is C₄-C₁₂ alkylene,such as an unsubstituted C₄-C₁₂ alkylene. More preferably, R⁶ is C₄-C₁₀,C₄-C₈, or C₆-C₈ alkylene, such as an unsubstituted C₄-C₁₀, C₄-C₈, orC₆-C₈ alkylene. According to one embodiment, one of R¹-R⁵ is hydroxy,for example, R¹ can be hydroxy.

According to yet another embodiment, when R⁶ is a C₁-C₁₀ alkylene, atmost one of R² and R⁴ is halogen. According to another embodiment, R⁶ isa C₈-C₁₆, C₉-C₁₆, C₁₀-C₁₆, or C₁₁-C₁₆ alkylene. For instance, R⁶ may bea C₈, C₉, C₁₀, C₁₁, or C₁₂ alkylene (e.g., a normal C₈-C₁₂ alkylene).According to yet another embodiment, at most one of R¹ and R⁵ is alkyl.

According to yet another embodiment, R¹ is hydroxy and R², R³, R⁴, andR⁵ are independently hydrogen or halogen.

According to yet another embodiment, R² is hydroxy and R¹, R³, R⁴, andR⁵ are independently hydrogen or halogen.

According to yet another embodiment, R³ is hydroxy and R¹, R², R⁴, andR⁵ are independently hydrogen or halogen.

In a preferred embodiment, halogen is F, Cl or Br, more preferably F orCl, and even more preferably Cl.

According to yet another embodiment, R⁶ is C₁-C₁₆ alkylene, (C₁-C₁₆alkyl) arylene or aryl(C₁-C₁₆ alkylene). More preferably R⁶ is C₁-C₁₂alkylene, more preferably C₃-C₁₀ alkylene, more preferably C₄-C₁₀ orC₄-C₈ alkylene, and more preferably C₆-C₈ alkylene. More preferably, R⁶is unsubstituted.

According to yet another embodiment, R⁷ is —NR¹⁸R¹⁹ and R¹⁸ and R¹⁹ areindependently C₁-C₄ alkyl (e.g., methyl, ethyl, propyl, or butyl)substituted with —OH. In another embodiment, R⁷ is —NR¹⁸R¹⁹ and R¹⁸ andR¹⁹ combine to form a six membered heterocyclic ring substituted with anoxo group.

According to one preferred embodiment, R¹ is hydrogen; R², R³, and R⁴are independently hydrogen, halogen, —OH, or —OCH₃; R⁵ is hydrogen, —OH,or —C(O)CH₃; R⁶ is C₁-C₁₂ alkylene, and R⁷ is NR¹⁸R¹⁹ wherein R¹⁸ andR¹⁹ combine to form a 5, 6 or 7 membered heterocyclic ring.

According to another preferred embodiment, one of R³, R⁴, and R⁵ ishydroxy and the others are independently halogen or hydrogen; R¹ and R²are independently halogen or hydrogen; R⁶ is C₁-C₁₆ alkylene; and R⁷ isNR¹⁸R¹⁹ wherein R¹⁸ and R¹⁹ combine to form a 5, 6, or 7 memberedheterocyclic ring. R⁶ is preferably C₆-C₁₆, C₆-C₁₀, C₈-C₁₆, C₁₀-C₁₆, orC₄-C₈ alkylene, such as unsubstituted C₆-C₁₆, C₆-C₁₀, C₈-C₁₆, C₁₀-C₁₆,or C₄-C₈ alkylene. Preferably, R¹⁸ and R¹⁹ form a morpholino orimidazole.

In another preferred embodiment, R¹ is hydrogen; R², R³, and R⁴ areindependently hydrogen, halogen, —OH, or —OCH₃; R⁵ is hydrogen, —OH, or—C(O)CH₃; R⁶ is C₁-C₁₂ alkylene; and R⁷ is N⁺R¹⁸R¹⁹R²⁰(Y⁻) wherein R¹⁸and R¹⁹ are hydroxy substituted C₁-C₁₆ alkyl and R²⁰ is hydrogen.

In another preferred embodiment, R¹ is hydrogen; R², R³, and R⁴ areindependently hydrogen, halogen, —OH, or —OCH₃; R⁵ is hydrogen, —OH, or—C(O)CH₃; R⁶ is C₁-C₁₂ alkylene; and R⁷ is N⁺R¹⁸R¹⁹R²⁰ (Y⁻) wherein R¹⁸and R¹⁹ are hydroxy substituted C₁-C₁₆ alkyl and R²⁰ is hydrogen.

In another preferred embodiment, R¹, R², R⁴, R⁵ are independentlyhalogen or hydrogen; R³ is —OH, or —OCH₃; and R⁷ is N⁺R¹⁸R¹⁹R²⁰ (Y⁻)wherein R¹⁸ and R¹⁹ are hydroxy substituted C₁-C₁₆ alkyl and R²⁰ ishydrogen.

According to one preferred embodiment, R¹ is hydrogen; R², R³, and R⁴are independently hydrogen, halogen, —OH, or —OCH₃; R⁵ is hydrogen, —OH,or —C(O)CH₃; R⁶ is C₁-C₆ alkylene or aryl substituted C₁-C₁₂ alkyl; andR⁷ is —NR¹⁸R¹⁹ wherein R¹⁸ and R¹⁹ combine to form a 5, 6, or 7 memberedheterocyclic ring or N⁺R¹⁸R¹⁹R²⁰ (Y⁻) wherein R¹⁸ and R¹⁹ are hydroxysubstituted C₁-C₁₆ alkyl and R²⁰ is hydrogen.

In another preferred embodiment, the citrate salt of the delivery agentis used.

Other suitable delivery agents include those having the followingstructure and pharmaceutically acceptable salts thereof:

wherein

R¹, R², R³, and R⁴ are independently H, —OH, halogen, —OCH₃, —NR¹⁰R¹¹ or—N⁺R¹⁰R¹¹R¹²(R¹³)⁻;

R⁵ is H, —OH, —NO₂, —NR¹⁴R¹⁵, —N⁺R¹⁴R¹⁵R¹⁶ (R¹³)⁻, amide, C₁-C₁₂ alkoxy,C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, carbamate, carbonate, urea, or —C(O)R¹⁸;

R⁵ is optionally substituted with —OH, —SH, or —COOH;

R⁵ is optionally interrupted by O, N, S, or —C(O)—;

R⁶ is a C₁-C₁₂ alkylene, C₁-C₁₂ alkenylene, or arylene;

R⁶ is optionally substituted with a C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄alkoxy, —OH, —SH, halogen, —NH₂, or —CO₂R⁹;

R⁶ is optionally interrupted by O or N;

R⁷ is a bond or arylene;

R⁷ is optionally substituted with —OH, halogen, —C(O)CH₃, —NR¹⁰R¹¹ or—N⁺R¹⁰R¹¹R¹²(R¹³)⁻;

R⁸ is H or C₁-C₄ alkyl;

R⁹ is H, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R¹⁰, R¹¹, and R¹² are independently H or C₁-C₁₀ alkyl;

R¹³ is a halide, hydroxide, sulfate, tetrafluoroborate, or phosphate;

R¹, R¹⁵, and R¹⁶ are independently H, C₁-C₁₀ alkyl, C₂-C₁₂ alkenyl, O,or —C(O)R¹⁷;

R¹⁷ is —OH, C₁-C₁₀ alkyl, or C₂-C₁₂ alkenyl; and

R¹⁸ is —OH, C₁-C₆ alkyl, —NR¹⁴R¹⁵, —N⁺R¹⁴R¹⁵R¹⁶ (R¹³)⁻.

According to one embodiment, when R⁵ is OCH₃ then R⁶ is C₁-C₈ or C₁₀-C₁₂alkyl.

According to a preferred embodiment, R⁵ is not —OCH₃. More preferably,R⁵ is not alkoxy.

According to another preferred embodiment, R¹, R², R³, and R⁴ arehydrogen, R⁵ is —COOH, —C(O)NH₂, —C(O)CH₃, or —NO₂, R⁶ is —(CH₂)₇—, andR⁷ is a bond.

According to yet another preferred embodiment, R¹, R², R³, and R⁴ arehydrogen, R⁵ is —C(O)NH₂, R⁶ is —CH₂—, and R⁷ is a para-phenylene.

According to one embodiment, the delivery agents of formula (6) have theformula:

wherein

R¹⁹ is —NO₂ or —C(O)R²³;

R²⁰ is a C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene;

R²¹ is a bond or arylene;

R²² is H or C₁-C₄ alkyl; and

R² is —OH, C₁-C₆ alkyl, or —NH₂.

The delivery agent compound can also be any of those described in U.S.Pat. Nos. 6,699,467, 6,663,898, 6,693,208, 6,693,073, 6,693,898,6,663,887, 6,646,162, 6,642,411, 6,627,228, 6,623,731, 6,610,329,6,558,706, 6,525,020, 6,461,643, 6,461,545, 6,440,929, 6,428,780,6,413,550, 6,399,798, 6,395,774, 6,391,303, 6,384,278, 6,375,983,6,358,504, 6,346,242, 6,344,213, 6,331,318, 6,313,088, 6,245,359,6,242,495, 6,221,367, 6,180,140, 6,100,298, 6,100,285, 6,099,856,6,090,958, 6,084,112, 6,071,510, 6,060,513, 6,051,561, 6,051,258,6,001,347, 5,990,166, 5,989,539, 5,976,569, 5,972,387, 5,965,121,5,962,710, 5,958,451, 5,955,503, 5,939,381, 5,935,601, 5,879,681,5,876,710, 5,866,536, 5,863,944, 5,840,340, 5,824,345, 5,820,881,5,811,127, 5,804,688, 5,792,451, 5,776,888, 5,773,647, 5,766,633,5,750,147, 5,714,167, 5,709,861, 5,693,338, 5,667,806, 5,650,386,5,643,957, 5,629,020, 5,601,846, 5,578,323, 5,541,155, 5,540,939,5,451,410, 5,447,728, 5,443,841, and 5,401,516; InternationalPublication Nos. WO94/23767, WO95/11690, WO95/28920, WO95/28838,WO96/10396, WO96/09813, WO96/12473, WO97/36480, WO 2004/4104018, WO2004080401, WO 2004062587, WO 2003/057650, WO 2003/057170, WO2003/045331, WO 2003/045306, WO 2003/026582, WO 2002/100338, WO2002/070438, WO 2002/069937, WO 02/20466, WO 02/19969, WO 02/16309, WO02/15959, WO 02/02509, WO 01/92206, WO 01/70219, WO 01/51454, WO01/44199, WO 01/34114, WO 01/32596, WO 01/32130, WO 00/07979, WO00/06534, WO 00/06184, WO 00/59863, WO 00/59480, WO 00/50386, WO00/48589, WO 00/47188, WO 00/46182, WO 00/40203, WO 99/16427, WO98/50341, WO 98/49135, WO 98/34632, WO 98/25589, WO 98/21951, WO97/47288, WO 97/31938, WO 97/10197, WO 96/40076, WO 96/40070, WO96/39835, WO 96/33699, WO 96/30036, WO 96/21464, WO 96/12475, and WO96/12474; and U.S. Published Application Nos. 20040110839, 20040106825,20040068013, 20040062773, 20040022856, 20030235612, 20030232085,20030225300, 20030198658, 20030133953, 20030078302, 20030072740,20030045579, 20030012817, 20030008900, 20020155993, 20020127202,20020120009, 20020119910, 20020102286, 20020065255, 20020052422,20020040061, 20020028250, 20020013497, 20020001591, 20010039258, and20010003001. Each of the above listed U.S. patents and U.S. andInternational published applications are herein incorporated byreference.

Non-limiting examples of delivery agent compounds includeN-(8-[2-hydroxybenzoyl]-amino)caprylic acid,N-(10-[2-hydroxybenzoyl]-amino)decanoic acid,8-(2-hydroxy-4-methoxybenzoylamino)octanoic acid,8-(2,6-dihydroxybenzoylamino)octanoic acid,8-(2-hydroxy-5-bromobenzoylamino)octanoic acid,8-(2-hydroxy-5-chlorobenzoylamino)octanoic acid,8-(2-hydroxy-5-iodobenzoylamino)octanoic acid,8-(2-hydroxy-5-methylbenzoylamino)octanoic acid,8-(2-hydroxy-5-fluorobenzoylamino)octanoic acid,8-(2-hydroxy-5-methoxybenzoylamino)octanoic acid,8-(3-hydroxyphenoxy)octanoic acid, 8-(4-hydroxyphenoxy)octanoic acid,6-(2-cyanophenoxy)hexanoic acid,8-(2-Hydroxyphenoxy)octyl-diethanolamine, 8-(4-hydroxyphenoxy)octanoate,8-(4-hydroxyphenoxy)octanoate,8-(2-hydroxy-4-methoxybenzoylamino)octanoic acid,8-(2-hydroxy-5-methoxybenzoylamino)-octanoic acid, and salts thereof.Preferred salts include, but are not limited to, pharmaceuticallyacceptable salts thereof such as the monosodium and disodium salts.

The delivery agent compounds may be in the form of the carboxylic acidor pharmaceutically acceptable salts thereof, such as sodium salts, andhydrates and solvates thereof. The salts may be mono- or multi-valentsalts, such as monosodium salts and disodium salts. The delivery agentcompounds may contain different counter ions chosen for example due totheir effect on modifying the dissolution profile of the carrier.

The delivery agent compounds may be prepared by methods known in theart, such as those discussed in the aforementioned publications (e.g.,International Publication Nos. WO 98/34632, WO 00/07979, WO 01/44199, WO01/32596, WO 02/20466, and WO 03/045306). SNAC, SNAD, and the free acidand other salts thereof may be prepared by methods known in the art,such as those described in U.S. Pat. Nos. 5,650,386 and 5,866,536.

Salts of the delivery agent compounds of the present invention may beprepared by methods known in the art. For example, sodium salts may beprepared by dissolving the delivery agent compound in ethanol and addingaqueous sodium hydroxide.

The delivery agent compound may be purified by recrystallization or byfractionation on one or more solid chromatographic supports, alone orlinked in tandem. Suitable recrystallization solvent systems include,but are not limited to, acetonitrile, methanol, and tetrahydrofuran.Fractionation may be performed on a suitable chromatographic supportsuch as alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase chromatography using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater or an appropriate buffer as the mobile phase. When anion exchangechromatography is performed, preferably a 0-500 mM sodium chloridegradient is employed.

Solutions Containing a Delivery Agent Compound and Insulin or an AnalogThereof

Some processes of the present invention involve the introduction of adelivery agent and insulin into an aqueous solution and obtaining apowder from this solution (e.g. by lyophilization or rotaryevaporation). Generally water is used as the solvent, although thesolvent can also comprise or consist essentially of other solvents whichdissolve the delivery agent (e.g. sodium 4-CNAB) and insulin. Forexample, ethanol, methanol, isopropyl alcohol, tetrahydrofuran, dioxane,butanol, acetone, 2-butanone, methyl tert-butyl ether, n-propanol,methylene chloride, and other similar low boiling point solvents couldbe used in lieu of, or in combination with, water.

Once obtained, the powder (which contains insulin and a delivery agentcompound) may be further processed (e.g. milled or granulated with otherexcipients) and compressed into tablets or filled into capsules.

Gelatin Based Pharmaceutical Formulations

Gelatin is a mixture of purified protein fractions that may be obtainedby partial hydrolysis of animal collagen by an acid or an alkaline. Theprocess of acid hydrolysis is referred to as Type A and that by alkalinehydrolysis is referred to as Type B. Gelatin is a linear polymer that iscomprised of amino acids which could result in a molecular weightranging from 15,000 to 250,000. As used herein, the term gelatinincludes acid and alkaline hydrolysates of animal collagen.

Gelatin may be applied in formulations of the present invention to servemany functions, such as a coating, a suspending agent, tablet binderand/or as a viscosity-increasing agent. Insulin/delivery agent tablets(e.g. insulin/sodium 4-CNAB) may be formulated at various concentrationsof gelatin and at various ratios of insulin and delivery agent.

In water, gelatin swells and softens and it can absorb between 5-10times its own weight of water. There are several hydrophilic natural andsynthetic polymers may be applied, in certain embodiments, in place ofgelatin. For example, (a) anionic polymers: alginic acid, dextransulfate, pectin; (b) cationic acid: chitosan, polylysine; (c)amphiphatic polymers: carboxylmethyl chitin, fibrin; (d) neutralpolymers such as dextran, agarose, pullulan.

As used herein, the term gelatin includes gelatin and gelatinalternatives disclosed in Remington's Pharmaceutical Sciences, 16^(th)ed., Mack Publishing Company, Easton, Pa. (1980), page 1245 and pages1576-1582, which is hereby incorporated by reference in its entirety.The term gelatin also includes compositions disclosed in U.S. Pat. No.6,090,915, U.S. Pat. No. 4,043,996, U.S. Pat. No. 4,064,008, U.S. Pat.No. 4,176,117, U.S. Pat. No. 4,889,920, U.S. Pat. No. 4,374,063, U.S.Pat. No. 5,210,182, U.S. Pat. No. 4,232,425, U.S. Pat. No. 4,402,873,U.S. Pat. No. 4,427,583, U.S. Pat. No. 5,093,474, U.S. Pat. No.5,288,408 and U.S. Pat. No. 5,459,241, each of which is herebyincorporated by reference in their entirety.

The term gelatin, as used herein also includes gelatin substitutes andalternatives. Generally, such a gelatin alternative can be made fromeasily obtainable (e.g. vegetable) materials having a homogeneouscomposition and having all the essential characteristics of gelatin. Inthe manufacture of soft gel films and capsules, the soft gel compositionpreferably possesses the properties of good wet and dry film strength,insolubility in cold water, oil, and alcohol, solubility in hot water,temperature and pressure sealability, film clarity, film flexibility,edibility, inertness to drugs or other materials to be encapsulated, andrapid setting from a hot liquid to form a gel.

One gelatin alternative is a film-forming composition that comprisesstarch material selected from modified starch and waxy starch; gum; andplasticizer as disclosed in U.S. Pat. No. 6,375,981, which is herebyincorporated by reference. The modified starch or waxy starch preferablyhas a dextrose equivalent (DE) of less than about 1, and more preferablyhas no measurable DE. This composition can be, but is not required tobe, 100% gelatin-free. Thus, the composition can be used as a gelatinreplacement, or as an extender in gelatin formulations.

Another gelatin alternative is wheat fiber gel as disclosed in U.S. Pat.No. 6,440,480, which is hereby incorporated by reference. Wheat fibergel is made by thermal/physical processing of wheat fiber. A specialmilling technique is used for treating wheat material resulting in aproduct containing a large proportion of microfine particles. Specificimprovements are obtained by mixing the product with maltodextrin. Theproduct so obtained is sold under the tradename Vitacel®, by FMCBiopolymer of Philadelphia, Pa. This product is a dry powder, whichreadily disperses in water. Upon stirring of the dispersion the gelforms through shear forces. It is reported that wheat fiber gel can beused as a gelatin replacer in yogurt or ice cream. (I. I. Bollinger,Food Marketing & Techn. October 1995, 4-6).

Carrageenan is yet another gelatin alternative. Carrageenan is a naturalhydrocolloid, a polysaccharide hydrocolloid, which is derived fromseaweed. It comprises a carbohydrate polymer of repeating sugar units,which is linear, without significant numbers of branches orsubstitutions.

Methods of Treatment

The present invention also provides methods for treating a subject withimpaired glucose tolerance or with early or late stage diabetes compriseorally administering to the mammal a pharmaceutical formulation of thepresent invention that includes a therapeutically effective amount ofinsulin or an insulin analog and a delivery agent in an amount effectiveto facilitate the absorption of the insulin.

The pharmaceutical formulations may also include a biguanide such asmetformin, as disclosed in International Application No. PCT/US05/27499,which is hereby incorporated by reference.

It is preferred that the administration be on a chronic basis, e.g., forat least two weeks. In various embodiments, the administration ispreprandially and at bedtime such that, after two weeks of treatment,the subject achieves improved glucose tolerance and glycemic control, aswell as improved insulin utilization, insulin sensitivity, insulinsecretion capacity and/or HbA₁c levels, as compared with baseline levelsprior to treatment.

Improved glucose tolerance and better endogenous capacity of the subjectto handle sugar load can also be measured by an AUC of blood glucoseexcursion, following a glucose load, that is reduced by a statisticallysignificant amount as compared with AUC of blood glucose excursion,following a glucose load, prior to treatment.

Improved glycemic control can be demonstrated by:

-   -   decreased fasting blood glucose levels as measured by fasting        blood glucose concentration that is reduced by a statistically        significant amount as compared with baseline fasting blood        glucose concentration prior to treatment.    -   decreased serum fructosamine concentrations, as measured by        serum fructosamine assay, that is reduced by a statistically        significant amount as compared with baseline serum fructosamine        concentrations prior to treatment.    -   improved HbA1c levels after treatment compared with baseline        levels prior to treatment. Preferably, the improved HbA1c levels        are measured by a statistically significant decline in HbA1c        levels. When treating a mammal with impaired glucose tolerance        or with early or late stage diabetes, administration of the        pharmaceutical formulation of the present invention can        preferably be made to a mammal having an HbA_(1c) level ranging        from normal to elevated prior to treatment.

Improved insulin utilization and insulin sensitivity of the subject'sbody can be measured by a statistically significant decline in HOMA(Homeostasis Model Assessment). Improved insulin secretion capacity ofthe subject's body may also be measured by Stumvoll first-phase insulinsecretion capacity index.

In preferred embodiments of the invention, by virtue of the chronicadministration of oral dosage forms of the present invention, thesubject achieves improved glucose tolerance and glycemic control ascompared with baseline levels prior to treatment even without anystatistically significant increase in weight, any statisticallysignificant increase in risk of hypoglycemia or any statisticallysignificant increase in risk of hyperinsulinemia in the mammal over thetreatment period, and without the need for monitoring the mammal's bloodglucose concentrations or HbA₁c levels. Further, by virtue of thechronic administration of oral dosage forms of the present invention,the subject achieves improved insulin utilization, insulin sensitivityinsulin secretion capacity and HbA₁c levels as compared with baselinelevels prior to treatment.

It is preferred that the administration of the oral pharmaceuticalformulation is administered 1-4 or more times daily, preprandiallyand/or at bedtime. In one embodiment of the invention, administration ofthe pharmaceutical formulation takes place once daily, either at bedtimeor preprandially for one meal during the day time, e.g., for breakfast,lunch or dinner. In another embodiment, administration of thepharmaceutical formulation takes place multiple times daily, preferablyat bedtime and preprandially for one meal during the day time, e.g., forbreakfast, lunch or dinner. In a further embodiment, administration ofthe pharmaceutical formulation takes place multiple times daily,preferably at bedtime and preprandially for more than one meal duringthe day time. Administration of the pharmaceutical formulation can alsobe at or shortly prior to bedtime and concurrently with or shortly priorto ingestion of each meal, i.e., within about 15 minutes or less ofingestion of each meal.

Preferably, the insulin formulations are administered to human patientson a chronic basis, e.g., for at least about two weeks. The dosage formof the present invention can be administered for at least one day, forone week, for two weeks, for longer periods, for alternating on-off timeperiods, or for the life of the patient.

The frequency of administration of the oral pharmaceutical formulation,on a daily basis (i.e., how often during one day-night period) and on achronic basis (i.e., for how many days), may depend upon the patient'sposition along a “diabetes continuum”, i.e., the extent of the patient'simpaired glucose tolerance, the patient's stage of diabetes and thepatient's need for exogenous glycemic control. This continuum rangesfrom normal glycemic control, to simple impaired glucose tolerance andinsulin resistance seen in pre-diabetics or early stage type 2diabetics, to failure of insulin production by the pancreas seen in type1 diabetics and late stage type 2 diabetics. This can also be measuredby the patient's HbA₁c concentration, ranging from normal to elevatedlevels (e.g., a HbA1c value of 8.0% or greater).

For example, if the subject has a need for fasting glycemic control, theoral pharmaceutical formulation should preferably be administered onlyat or shortly prior to bedtime. If the subject has some need forpost-prandial glycemic control, the oral pharmaceutical formulationshould preferably be administered preprandially for some meals. If thesubject has a need for total post-prandial glycemic control, the oralpharmaceutical formulation should preferably be administeredpreprandially for all meals. If the subject has a need for comprehensiveglycemic control, the oral pharmaceutical formulation should preferablybe administered preprandially for all meals and at or shortly prior tobedtime.

Embodiments of the present invention also provide a method of achievingglucose homeostasis in subjects, comprising orally administering to asubject a pharmaceutical formulation comprising a therapeuticallyeffective amount of insulin or an insulin analog and a delivery agent inan amount effective to facilitate the absorption of the insulin orinsulin analog. It is preferred that the administration be on a chronicbasis, e.g., for at least two weeks, and be preprandially and at bedtimesuch that, after two weeks of treatment, the mammal achieves improvedglucose tolerance and glycemic control as compared with baseline levelsprior to treatment.

EXAMPLES

The following examples illustrate the invention without limitation. Allparts are given by weight unless otherwise indicated.

Example 1—Solubilization of Insulin

The solubility of recombinant human zinc insulin obtained from DiosynthFrance (distributed in the U.S. through Diosynth, Inc.) (hereafterinsulin type #1) and Eli Lilly Co. (hereafter insulin type #2) wasdetermined in aqueous solutions having various pH values at 37° C.Excess insulin was added to buffer solutions at pH values of 1, 2, 3, 4,5, 6, 6.8, and 7.4. Vials containing the solutions were shaken in aconstant temperature water bath, and visually observed at regular timeintervals. Samples were taken at 2 hours, 4 hours, and 24 hours andanalyzed by a stability-indicating HPLC method. The results are shownbelow in Table 1:

TABLE 1 pH-solubility data of Diosynth and Lilly insulin at 37° C.Insulin concentration Insulin concentration Insulin concentration(mg/ml), 2 h (mg/ml), 4 h (mg/ml), 24 h insulin insulin insulin insulininsulin insulin pH type # 1 type # 2 type # 1 type # 2 type # 1 type # 21.23  >2, deg* >2, deg   >4, deg   >4, deg 0.814, deg deg 2.02 >2,deg >2, deg   —, deg   —, deg   >4, deg   >2, deg 3.02 0.989, deg  0.797, deg   1.092, deg 0.814, deg 0.826, deg 0.731, deg 4.05 0.0660.068 0.0317 0.040 0.147, deg 0.128, deg 5.07 0.156 0.091 0.155  0.0940.165, deg 0.124, deg 6.08 0.066 0.049 0.0612 0.044 0.126, deg 0.229,deg 6.82 1.852 0.947 >2     1.294 >4 >27.42 >2    >2    >4     >4    >4 >4 deg - products of degradationpresent in chromatograms

Visual observation indicated that insulin type #1 dissolved faster thaninsulin type #2. This difference was not observed at pH 1, 2, and 7.4where dissolution was very rapid for both insulin types. Solubility atpH 1, 2 and 7.4 was in excess of 4 mg/mL. Solubility at pH 6.8 wasgreater than 4 mg/mL for insulin type #1 and 2 mg/mL for insulin type#2, but complete dissolution occurred only after overnight shaking. Thedifference in the dissolution rate of insulin type #2 was most apparentat this pH. Both types of insulin exhibited some degradation at pH 3, 4,5, and 6 after 24 hours of shaking. As expected, solubility was lowestaround pH 5, with the solubilities at pH 4, 5, and 6 all being around0.1 mg/mL.

The results for pH 3-6.8 are shown in FIG. 1.

Example 2—Solubilization of Insulin in Solutions Containing a DeliveryAgent

The solubility of insulin was investigated in aqueous solutionscontaining varying amounts of the delivery agent compound sodium 4-CNAB.The aqueous solution containing delivery agent was adjusted to pH 1 bythe addition of 1N HCl and excess insulin was added to the acidifiedsolution. Thereafter, the pH of the solution was increased by theaddition of increments of 1N NaOH. Vials containing the suspensions wereleft without shaking or sonicating for 1-2 hours. The supernatant wasanalyzed for insulin and sodium 4-CNAB using an HPLC method and the pHwas recorded. This process was repeated with increased amounts ofdelivery agent added to the solution until the insulin was completelysolubilized. All of these experiments were performed at roomtemperature. The results are set forth below in Table 2.

TABLE 2 pH of Delivery Delivery Agent, Insulin, Delivery Agent Agentsolution (mg/mL) (mg/mL) None (DI water) 6.3 — 1.14 4-CNAB 6.4 5 0.56.45 10 1 6.26 50 11 6.49 50 44 6.71 100 86 6.45 100 >110 6.83 150 1876.79 200 >135 SNAC 7.9 150 >200 HPOD 3.9 250 >50

The results for 4-CNAB and Insulin are also shown in FIG. 2.

Example 4—Solubilization of Insulin Tablet

A 300 mg pellet of insulin was prepared in a die. The surface area ofthe pellet available to the dissolution medium was 0.484 cm². The pelletwas compressed at 1200-1400 lbs on a Carver press to form discs. The diewas then attached to the shaft of a dissolution apparatus (USPDissolution Type II (Paddle) made by Vankel). The die was rotated at 100rpm and then immersed in 500 mL of degassed dissolution mediummaintained at 37° C. Dissolution experiments were conducted in water andin aqueous solutions containing sodium 4-CNAB. Samples of the solutionswere taken over two hours and analyzed by HPLC. FIGS. 3 and 4 showdissolution profiles of insulin in deionized (DI) water and in 10 mg/mLsodium 4-CNAB solution, respectively.

The rate of insulin dissolution was significantly greater in the 10mg/mL sodium 4-CNAB dissolution media than the deionized waterdissolution media.

The experiments were repeated in 50 mg/ml sodium 4-CNAB dissolutionmedia. The insulin levels in these solutions were below the detectionlimits of the HPLC technique used.

Example 6—Lyophilized Insulin/4-CNAB Formulations

Lyophilization as a method of co-drying insulin/4-CNAB solutions toobtain co-dried insulin/sodium 4-CNAB powder was investigated. The 3formulations shown in Table 5 were prepared as follows.

Initially, 4-CNAB was used to solubilize the insulin. The requiredamounts of insulin and sodium 4-CNAB were weighed out. The sodium 4-CNABwas added to the required amount of water (about 20 ml of water per gramof sodium 4-CNAB) and stirred (1-5 minutes) until completely dissolved.The corresponding amount of insulin was then dispersed in the sodium4-CNAB solution and left without stirring, shaking or sonicating for0.5-2 hours until solution became clear. The solution was lyophilizedusing the cycle in shown in Table 4.

TABLE 4 The freeze-drying cycle for Insulin/4-CNAB solutions.Temperature ° C. Time (minutes) Pressure (Torr) −10 30 −45 30 500 −351200 100 −35 240 50 25 1440 25

TABLE 5 Components Formulation 1 Formulation 2 Formulation 3 RecombinantHuman 1.8 mg   1.8 mg  1.8 mg Insulin (50 Units) (Strength: 27.5Units/mg) 4-CNAB Monosodium 80 mg 160 mg  240 mg Salt Dibasic Calcium 36mg  46 mg 79.75 mg  Phosphate Magnesium stearate 1.2 mg   2.2 mg 3.55 mgTotal Weight/tablet 119 mg  210 mg  325 mg

Tablets were prepared by mixing the lyophilized insulin/4CNAB powder,dibasic calcium phosphate and magnesium stearate. The powder mixture wascompressed into tablets using a single punch Korsch EK-0 tablet press toprepare an initial compact. Granules were obtained by crushing theinitial compact in a mortar and passing the granules through a 35 meshsieve. The granules were compressed into tablets of a predeterminedweight and stored in a freezer at −20° C.

Example 7—In-Vivo Primate Studies

The formulations of Example 6 were fed to rhesus monkeys. The monkeyswere fasted for at least 12 hrs prior to dosing and up to 4 hrs afterdosing. Water was withheld approximately 1 hr before dosing and up to 2hrs after dosing after which it was permitted ad libitum. The dosing wasfollowed by a 5 ml water flush. Blood samples (approximately 2 ml each)were collected by venipuncture at 15 minutes before dosing (t=0) and at5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 2, 3, 4 hr after dosing.

Four male primates were administered one tablet of Formulation 1 ofExample 6. The results are shown in FIG. 5 (glucose reduction) and FIG.6 (serum insulin concentrations). The averaged results are shown in FIG.7. After a wash-out period, the same group of four primates were eachadministered Formulation 2 of Example 6. These results are shown inFIGS. 8-10.

A different group of four male primates were each administered onetablet of Formulation 3 of Example 6. Glucose reduction is shown in FIG.11. Serum insulin levels for 3 of the four monkeys are shown in FIG. 12.

Example 8—Preparation of Tablets Containing Co-LyophilizedInsulin/4-CNAB Powder

Tablets containing co-lyophilized insulin/sodium 4-CNAB powder andhaving the formulation shown in Table 6 were prepared as follows. First,insulin was dissolved in an aqueous solution containing sodium 4-CNABand the solution was freeze dried according to the regimen in Table 4.The insulin/4-CNAB solution obtained from freeze-drying was then milledwith a 35 mesh sieve and blended with copovidone and magnesium stearate(intragranular excipients). The composition was then dry granulated byroller compaction. Copovidone and magnesium stearate was addedextragranularly and the granules were compressed into tablets at apressure of about 1000 psi for 5 seconds.

Based on the process, tablets with the following amounts of ingredientswere prepared:

TABLE 6 Insulin/4-CNAB (150 units/240 mg) Co-lyophilized Tablets WeightWeight/ Ingredients (mg/tablet) Batch (g) Insulin/4-CNAB (Co-lyophilized5.45/240 270.0 containing 150 Units of insulin) Copovidone; NF/EP(intragranular) 3.60 3.96 Magnesium Stearate; 0.90 0.99 NF/EP(intragranular) Copovidone; NF/EP (extragranular) 3.60 3.96 DibasicCalcium Phosphate 103.75 114.13 Anhydrous, USP/EP Magnesium Stearate2.70 2.97 Theoretical Tablet Weight 360 mg

Example 9 Preparation of Tablets Containing Co-LyophilizedInsulin/4-CNAB Powder

Tablets containing co-lyophilized insulin/4-CNAB and having theformulation shown in Table 7 were prepared by the procedure set forth inExample 8.

TABLE 7 Insulin/4-CNAB (150 units/400 mg) Co-lyophilized Tablets WeightWeight/ Ingredients (mg/tablet) Batch (g) *Insulin/4-CNAB(Co-lyophilized) 5.45/400 430.588 (Containing 150 units of Insulin)Copovidone; NF/EP (intragranular) 5.80 6.160 Magnesium Stearate; NF/EP(intragranular) 1.40 1.487 Copovidone; NF/EP (extragranular) 5.75 6.106Dibasic Calcium Phosphate Anhydrous, 157.20 166.946 USP/EP MagnesiumStearate 4.40 4.673 Theoretical Tablet Weight 580 mg

Example 10—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

Sodium 4-CNAB (935.7 g, 0.4 wt % water by Karl Fisher titration) andpurified water (4850 mL) were charged to a 20 L, borosilicate, rotaryevaporator flask. The flask was attached to a rotary evaporator androtated at about 60 rpm until the solids dissolved. The rotation wasstopped. The flask was removed from the rotary evaporator and insulin(64.1 g, 27.4 U/mg) was added. The flask was re-attached to the rotaryevaporator and the insulin was allowed to dissolve without agitation.The water was removed rapidly with the rotary evaporator bath set atabout 45 C and the internal pressure set at about 5 mm Hg. The walls ofthe rotary evaporator flask became coated with solid co-driedinsulin/4CNAB as the water was removed. These solids were scraped fromthe walls of the flask and dried in a vacuum oven set at full vacuum and50° C. until the water content was less than 10 wt % by Karl Fisheranalysis. In most cases the vacuum drying time could be reduced bybreaking up the larger lumps of co-dried material about midway throughthe vacuum oven drying cycle. The dried material was then hammer milledso that it would pass through a 35 mesh screen. The milled, powderymaterial was placed in a suitable container and stored in a freezer at−20° C. or lower until used. The recovery of co-dried material was about95%.

The co-dried insulin/4-CNAB prepared as described above, was mixed withpovidone and magnesium stearate in the amounts shown in Table 8 belowand compressed into tablets at a pressure of about 1000 psi for 5seconds.

TABLE 8 Ingredient Amount 4-CNAB Sodium Salt 80 mg  Recombinant Human150 Units (27.4 U/mg) Insulin Povidone, USP 1 mg (Kollidon 90F)Magnesium stearate, NF 1 mg

Example 11—Preparation of Capsules Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder. 80 mg of sodium 4-CNAB/150 Units of insulin wasplaced, without excipients, into size 2 hard gelatin opaque whitecapsules.

Example 12—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 966.7 g of 4-CNAB, 5000 mL of waterand 33.23 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 9 were prepared by theprocedure set forth in Example 10:

TABLE 9 Ingredient Amount 4-CNAB Sodium Salt 80 mg  Recombinant Human 75Units (27.4 U/mg) Insulin Povidone, USP 1 mg (Kollidon 90F) Magnesiumstearate, NF 1 mg

Example 13—Preparation of Capsules Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 966.7 g of 4-CNAB, 5000 mL of waterand 33.23 g of insulin were charged to the rotary evaporator flask.

80 mg of sodium 4-CNAB/75 Units of insulin was placed, withoutexcipients, into size 2 hard gelatin opaque white capsules.

Example 14—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of Example 10 was repeated to prepare tablets, except thatpovidone and magnesium stearate were not included in the formulation.

Example 15 Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of Example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 241.7 g of 4-CNAB, 1260 mL of waterand 8.3 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 10 were prepared by theprocedure set forth in Example 10.

TABLE 10 Ingredient Amount 4-CNAB Sodium Salt 160 mg  Recombinant Human150 Units (27.4 U/mg) Insulin Povidone, USP 0 mg (Kollidon 90F)Magnesium stearate, NF 0 mg

Example 16—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 245.8 g of 4-CNAB, 1280 mL of waterand 4.3 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 11 were prepared by theprocedure set forth in Example 10.

TABLE 11 Ingredient Amount 4-CNAB Sodium Salt 320 mg  Recombinant Human150 Units (27.4 U/mg) Insulin Povidone, USP 0 mg (Kollidon 90F)Magnesium stearate, NF 0 mg

Example 17—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 245.4 g of 4-CNAB, 1280 mL of waterand 5.6 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 12 were prepared by theprocedure set forth in Example 10.

TABLE 12 Ingredient Amount 4-CNAB Sodium Salt 160 mg  Recombinant Human100 Units (27.4 U/mg) Insulin Povidone, USP 0 mg (Kollidon 90F)Magnesium stearate, NF 0 mg

Example 18—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained FROM Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 247.2 g of 4-CNAB, 1300 mL of waterand 2.8 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 13 were prepared by theprocedure set forth in Example 10.

TABLE 13 Ingredient Amount 4-CNAB Sodium Salt 320 mg  Recombinant Human100 Units (27.4 U/mg) Insulin Povidone, USP 0 mg (Kollidon 90F)Magnesium stearate, NF 0 mg

Example 19—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained from Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 247.2 g of 4-CNAB, 1300 mL of waterand 2.8 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 14 were prepared by theprocedure set forth in Example 10:

TABLE 14 Ingredient Amount 4-CNAB Sodium Salt 320 mg  Recombinant Human100 Units (27.4 U/mg) Insulin Povidone, USP 1 mg (Kollidon 90F)Magnesium stearate, NF 1 mg

Example 20—Preparation of Tablets Containing Co-Dried Insulin/4-CNABPowder Obtained FROM Rotary Evaporation

The process of example 10 was repeated to prepare the co-driedinsulin/4-CNAB powder, except that 245.8 g of 4-CNAB, 1280 mL of waterand 4.3 g of insulin were charged to the rotary evaporator flask.

Tablets having the formulation shown in Table 15 were prepared by theprocedure set forth in Example 10.

TABLE 15 Ingredient Amount 4-CNAB Sodium Salt 320 mg  Recombinant Human150 Units (27.4 U/mg) Insulin Povidone, USP 1 mg (Kollidon 90F)Magnesium stearate, NF 1 mg

Example 21—Preparation of Tablets Based on Granulated Gelatin-BasedFormulation

Tablets having the formulation shown in Table 16 were prepared asfollows. Gelatin was milled using a Kitchen-Aid™ coffee grinder andlater screened through a sieve of size #35. Afterwards, the requiredamount of insulin was weighed and dispersed into purified water. Theamount of purified water used was about 15% of the theoretical batchweight. For a theoretical batch size of 1200 tablets, 50 g of Insulin isdispersed in 46.7 g of purified water.

Sodium 4-CNAB and the milled gelatin were transferred into anappropriate sized high shear granulator and mixed for about 2 minutes.The sodium 4-CNAB and gelatin mixture was granulated first with theaqueous dispersion of insulin and later with purified water using a pumpdevice. The resultant wet granules were evenly dispersed in oven traysand dried in a vacuum oven (Temperature=50° C.; Vacuum=5 mm Hg) for atleast 8 hours. The dried granules were characterized based on moisture(0.5%) and insulin content and insulin content uniformity. The granuleswere milled and screened through a sieve of size 0.02 inches. Prior totablet compression, dibasic calcium phosphate and magnesium stearatewere blended with dry granules. Tablets were compressed using an EKOsingle punch station press.

Based on the above procedure, tablets containing the following amountsof ingredients were prepared.

TABLE 16 Weight Ingredients (mg/dose) Recombinant Human Insulin 5.454-CNAB Monosodium salt 240 Gelatin (Type A) 12 Dibasic Calcium Phosphate(extragranular) 113.80 Magnesium Stearate (extragranular) 3.75 TotalWeight (mg/tablet) 375

The tablets prepared by this process had an average weight of about373.5 mg, a thickness of 5 mm and an average hardness of about 10.3 kP.

Example 22—Preparation of Granulated Tablets (Wet Granulation)

Tablets having the formulation shown in Tables 17 and 18 were preparedas follows.

TABLE 17 Drug Product Components: Insulin/4-CNAB (150 Units/80 mg)Tablets Component Function 4-CNAB Sodium Salt Delivery Agent RecombinantHuman Insulin Drug (Active Agent) Povidone, USP (Kollidon 90F) BinderDibasic Calcium Phosphate, Anhydrous, Binder USP Magnesium stearate, NFLubricant Purified Water, USP Granulating fluid

Sodium 4-CNAB was milled using a Quadro Comil equipped with a 35 meshscreen. Insulin and the milled sodium 4-CNAB were blended together, andtransferred to a Key Instruments KG 5 high shear granulator equippedwith a 5 Liter bowl. The material was granulated with povidone. Once theaddition of the povidone was completed, the container was rinsed withsmall portions of purified water and added to the granulation until thedesired granulation was achieved.

The granulation was transferred to clean stainless steel trays and driedin a vacuum oven at 50° C. until the moisture content was less than 5.0%w/w and then milled through a 35 mesh screen and further dried until themoisture content less than 1.5% w/w. The granulation was assayed forinsulin and sodium 4-CNAB using a validated HPLC method. The insulinassay of the granulation was used for calculating the required quantityof Emcompress for the batch. The required amount of Emcompress was addedto the granulation and blending was performed in a V-blender for 15minutes. Samples were collected for bend uniformity testing. Afteracceptable blend uniformity data was obtained the required amount ofmagnesium stearate was added and blending was performed for 3 minutes.The resulting blend was compressed into tablets using a Korsch EKOsingle station tablet press. The target tablet weight was 125 mg with arange of 119-131 mg, acceptable tablet hardness range was 5-11 kP with atarget tablet hardness of 7.0 kP. The tablets exhibited an averagethickness of 7.8 mm. The Insulin/4-CNAB (150 Units/80 mg) tablets werepackaged in a container closure system consisting of a 60 cc HDPE Round,White bottle with 33 mm Child Resistant Cap 1 with Safe-Gard® 75 mInduction Innerseal and Cotton Coil 12 gm/yd.

Based on this procedure, the following amounts of ingredients were usedto prepare the tablets:

TABLE 18 Drug Product Composition: Insulin/4-CNAB (150 Units/80 mg)Tablets (Batch Size 6,200 Tablets) Batch Component mg/Tablet Formula (g)4-CNAB Sodium Salt 76-84 496.0 Recombinant Human Insulin 5.32-5.88(142.5-157.5 35.1 Units) Povidone USP (Kollidon 90F) 0.38-0.42 2.2Dibasic Calcium Phosphate, 35.9-39.7 195.2 Anhydrous, USP MagnesiumStearate, NF 1.14-1.26 6.7 Impalpable Powder Total Weight (mg)118.8-131.3 735.2

Example 23—Human Clinical Study of Orally Administered Insulin

Six healthy male subjects between the ages of 18 and 40 were orallyadministered one tablet or capsule, depending on the treatment periodindicated below after an 8-hour fast the previous night. Glucose andinsulin values were obtained from blood samples fifteen minutes prior todosing (t=0) and 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 90 and 120minutes after dosing. After receiving the first tablet or capsule, thesubject underwent at least a 72 hour washout period before receiving thenext tablet or capsule.

The administration regimen was as follows:

TABLE 19 Treatment Period Summary of Dosage Preparation Details A Wetgranulation tablet Example 22 B Rotary evaporation tablet Example 10 CRotary evaporation capsule Example 11 D Rotary evaporation tabletExample 12 E Rotary evaporation capsule Example 13

Results for the treatment regiment are set forth below:

TABLE 20 Insulin Mean PK Results obtained from IndividualBaseline-adjusted Insulin Concentrations Cmax Tmax AUClast New/ReferenceNew/Reference Treatment (μU/mL) (min) (μU/mL*min) Cmax ratio AUC ratio AN 6 6 6 N/A N/A Mean 15.250 19.167 346.250 SD 10.064 2.041 227.404 Min4.50 15.00 40.00 Max 31.00 20.00 700.00 CV % 66.0 10.6 65.7 B N 6 6 61.8 1.2 Mean 27.500 19.167 422.708 SD 9.925 10.685 256.758 Min 14.5010.00 133.75 Max 41.50 40.00 860.00 CV % 36.1 55.7 60.7 C N 5 5 5 Mean8.800 35.000 260.000 SD 7.497 33.727 313.358 Min 0.50 10.00 11.25 Max21.00 90.00 805.00 CV % 85.2 96.4 120.5 C N 5 5 5 0.2 0.4 (without Mean5.200 41.000 128.000 outlier- SD 3.347 46.287 73.912 Subject 4, Min 0.5010.00 11.25 90 min Max 8.50 120.00 200.00 timepoint) CV % 64.4 112.957.7 D N 6 6 6 0.3 0.3 Mean 9.333 10.000 110.000 SD 4.215 3.162 59.713Min 1.00 5.00 7.50 Max 12.50 15.00 172.50 CV % 45.2 31.6 54.3 E N 5 4 50.4 0.4 Mean 5.600 38.750 140.500 SD 6.004 37.053 123.392 Min 0.00 5.000.00 Max 15.50 90.00 273.75 CV % 107.2 95.6 87.8

TABLE 21 Insulin Mean PK Results obtained from Individual InsulinConcentrations Cmax Tmax AUClast New/Reference New/Reference Treatment(μU/mL) (min) (μU/mL*min) Cmax ratio AUC ratio A N 6 6 6 N/A N/A Mean30.333 19.167 1882.917 SD 11.343 2.041 435.067 Min 16.00 15.00 1107.50Max 44.00 20.00 2222.50 CV % 37.4 10.6 23.1 B N 6 6 6 1.6 1.2 Mean47.167 19.167 2286.250 SD 14.148 10.685 1117.921 Min 21.00 10.00 747.50Max 59.00 40.00 4222.50 CV % 30.0 55.7 48.9 C(*) N 6 6 6 0.6 0.8 Mean18.667 27.500 1562.083 SD 4.179 45.689 247.454 Min 12.00 0.00 1127.50Max 24.00 120.00 1777.50 CV % 22.4 166.1 15.8 D N 6 6 6 0.8 0.9 Mean24.500 10.000 1640.417 SD 6.863 3.162 522.110 Min 18.00 5.00 867.50 Max36.00 15.00 2500.00 CV % 28.0 31.6 31.8 E N 6 6 6 0.9 0.9 Mean 26.16726.667 1744.167 SD 15.052 34.303 414.203 Min 16.00 0.00 1102.50 Max54.00 90.00 2222.50 CV % 57.5 128.6 23.7 (*)Insulin concentrationmeasured at 90 min in Subject 4 was considered an outlier hence notincluded in the PK analysis.

Model-independent pharmacokinetic metrics were calculated usingWinNonlin (v 4.5, Scientific Consulting, Inc.) from individual plasmaconcentration data. This program analyzes data using the standardmethods described by Gibaldi and Perrier. The area under the plasmaconcentration-time curve (AUC) was estimated by the linear trapezoidalrule.

“Baseline’ is defined as average plasma insulin concentrations asassessed prior to study drug administration (i.e. −15 and 0 minutesamples averaged). Adjusted concentrations were obtained by subtractingthe individual baseline from each individual time point (Ct−Co).Negative values were not included in the data analysis.

Results for the adjusted-mean insulin profiles are shown in FIGS. 13(without outlier) and 14 (with outlier).

Example 24—Human Clinical Study of Orally Administered Insulin

The protocol used in Example 23 was used for the following treatmentregimens:

TABLE 22 Treatment Period Summary of Dosage Preparation Details A RotaryEvaporation Tablets Example 10 B Rotary Evaporation Tablets Example 14 CRotary Evaporation Tablets Example 15 D Rotary Evaporation TabletsExample 16 E Rotary Evaporation Tablets Example 17 F Rotary EvaporationTablets Example 18

Results for the treatment regiment are set forth below:

TABLE 23 Insulin Mean PK Results obtained from IndividualBaseline-adjusted Insulin Concentrations Cmax Tmax AUClast Treatment(μU/mL) (min) (μU/mL * min) A N 6 6 6 Mean 30.633 17.500 795.615 SD20.977 5.244 542.094 Min 10.86 10.00 331.76 Max 66.62 25.00 1742.30 CV %68.5 30.0 68.1 B N 6 6 6 Mean 22.441 13.333 312.535 SD 16.489 2.582214.398 Min 8.00 10.00 118.83 Max 52.00 15.00 649.80 CV % 73.5 19.4 68.6C N 6 6 6 Mean 28.546 21.667 741.708 SD 23.848 14.024 737.074 Min 6.055.00 100.93 Max 69.20 45.00 1787.75 CV % 83.5 64.7 99.4 D N 6 6 6 Mean29.421 16.667 575.435 SD 17.155 4.082 384.295 Min 13.56 10.00 176.14 Max53.08 20.00 1210.13 CV % 58.3 24.5 66.8 E N 6 6 6 Mean 18.556 15.833303.702 SD 14.849 2.041 304.338 Min 1.49 15.00 13.35 Max 39.25 20.00799.38 CV % 80.0 12.9 100.2 F N 6 6 6 Mean 20.971 19.167 536.952 SD16.710 9.704 528.919 Min 2.14 5.00 15.06 Max 44.11 30.00 1312.61 CV %79.7 50.6 98.5

TABLE 24 Insulin Mean PK Results obtained from Individual InsulinConcentrations Cmax Tmax AUClast Treatment (μU/mL) (min) (μU/mL * min) AN 6 6 6 Mean 41.945 17.500 1905.513 SD 23.681 5.244 880.650 Min 14.9410.00 794.20 Max 81.36 25.00 3251.53 CV % 56.5 30.0 46.2 B N 6 6 6 Mean35.740 13.333 1590.567 SD 14.956 2.582 246.447 Min 20.30 10.00 1199.00Max 63.19 15.00 1961.45 CV % 41.8 19.4 15.5 C N 6 6 6 Mean 40.807 21.6671955.696 SD 24.954 14.024 896.595 Min 15.60 5.00 1032.25 Max 84.17 45.003295.53 CV % 61.2 64.7 45.8 D N 6 6 6 Mean 42.480 16.667 1919.300 SD18.411 4.082 493.982 Min 26.08 10.00 1363.75 Max 68.64 20.00 2586.25 CV% 43.3 24.5 25.7 E N 6 6 6 Mean 31.770 15.833 1563.100 SD 13.306 2.041334.769 Min 13.95 15.00 1030.88 Max 49.90 20.00 1984.25 CV % 41.9 12.921.4 F N 6 6 6 Mean 33.737 19.167 1861.629 SD 16.049 9.704 654.066 Min17.02 5.00 1271.05 Max 53.39 30.00 2969.95 CV % 47.6 50.6 35.1Results based on the adjusted mean insulin profiles are set forth inFIG. 15

Example 25—Human Clinical Study of Orally Administered Insulin

The protocol used in Example 23 was used for the following treatmentregimens:

TABLE 25 Treatment Period Summary of Dosage Preparation Details AGelatin-Based Tablet Example 21 B Co-lyophilized Tablets Example 8 CRotary Evaporation Tablets Example 19 D Rotary Evaporation TabletsExample 20 E Wet granulation Tablets Example 22 F Co-lyophilized TabletsExample 9

Results for the treatment regiment are set forth below:

TABLE 26 Insulin Mean PK Results obtained from IndividualBaseline-adjusted Insulin Concentrations Cmax Tmax AUClast New/ReferenceNew/Reference Treatment (μU/mL) (min) (μU/mL*min) Cmax ratio AUC ratio AN 6 6 6 2.6 3.6 Mean 30.633 17.500 764.871 SD 20.977 5.244 508.089 Min10.86 10.00 331.76 Max 66.62 25.00 1629.01 CV % 68.5 30.0 66.4 B N 6 6 60.9 1.3 Mean 10.531 32.500 286.569 SD 12.685 43.215 284.348 Min 1.3310.00 12.68 Max 33.61 120.00 731.20 CV % 120.5 133.0 99.2 C N 6 6 6 1.01.2 Mean 12.203 15.000 254.679 SD 11.976 3.162 256.555 Min 4.89 10.0050.65 Max 36.15 20.00 755.21 CV % 98.1 21.1 100.7 D N 6 6 6 1.6 1.9 Mean19.013 13.333 415.721 SD 24.735 6.055 601.162 Min 2.12 5.00 9.80 Max67.82 20.00 1576.64 CV % 130.1 45.4 144.6 E N 6 6 6 N/A N/A Mean 11.69317.500 213.752 SD 10.033 8.216 180.716 Min 0.69 10.00 1.73 Max 26.7030.00 415.56 CV % 85.8 46.9 84.5 F N 6 6 6 0.8 0.7 Mean 9.092 15.000142.221 SD 5.539 8.367 77.332 Min 1.83 10.00 4.56 Max 18.54 30.00 232.71CV % 60.9 55.8 54.4

TABLE 27 Insulin Mean PK Results obtained from Individual InsulinConcentrations Cmax Tmax AUClast New/Reference New/Reference Treatment(μU/mL) (min) (μU/mL*min) Cmax ratio AUC ratio A N 6 6 6 1.4 1.6 Mean32.098 36.667 1966.079 SD 13.956 40.947 963.466 Min 9.85 15.00 800.78Max 47.49 120.00 3345.18 CV % 43.5 111.7 49.0 B N 6 6 6 1.0 1.1 Mean21.730 32.500 1379.888 SD 10.133 43.215 281.651 Min 14.06 10.00 1151.90Max 38.54 120.00 1922.78 CV % 46.6 133.0 20.4 C N 6 6 6 1.1 1.2 Mean23.873 15.000 1437.488 SD 12.681 3.162 401.442 Min 12.92 10.00 927.30Max 48.83 20.00 2129.28 CV % 53.1 21.1 27.9 D N 6 6 6 1.3 1.2 Mean29.630 13.333 1492.971 SD 25.993 6.055 895.031 Min 7.20 5.00 398.93 Max78.99 20.00 2857.18 CV % 87.7 45.4 59.9 E N 6 6 6 N/A N/A Mean 22.50717.500 1239.967 SD 8.105 8.216 244.282 Min 14.67 10.00 878.80 Max 32.7030.00 1507.50 CV % 36.0 46.9 19.7 F N 6 6 6 0.8 0.8 Mean 18.093 15.0001030.767 SD 6.660 8.367 287.163 Min 11.84 10.00 648.45 Max 29.57 30.001360.88 CV % 36.8 55.8 27.9

Results based on the adjusted mean insulin profiles are set forth inFIG. 16

Example 26—Human Clinical Study of Orally Administered Insulin

145 humans patients having type 2 diabetes who were failing metforminmonotherapy were enrolled in a 90 day randomized double-blindplacebo-controlled study. The patients remained on their metforminregimen, which varied from 125 mg-3000 mg per day individualized foreach patient. 141 patients completed the study.

The patients were separated into 4 treatment groups as follows:

Group Insulin Dose Dosing Regimen 1 (n = 35) Tablet of Example 22 2insulin tablets, 4 times daily (150 IU of insulin each) 2 (n = 35)Tablet of Example 22 2 insulin tablets, 2 times daily (150 IU ofinsulin) 2 placebo tablets, 2 times daily 3 (n = 36) Tablet of Example22 1 insulin tablet + 1 placebo (150 IU of insulin) tablet, 4 timesdaily 4 (n = 35) Placebo 2 placebo tablets, 4 times daily

Hemoglobin A1c counts were obtained about three weeks prior to beginningthe study (screening) and just prior to the first administration of theoral insulin (t=0) or (baseline).

Changes in HbA1c levels at the conclusion of the 90 day study for thefour groups (relative to baseline) are shown in FIG. 17.

FIGS. 18 and 19 shows the difference between Hemoglobin A1c level atscreening and at baseline for each of the subjects.

FIG. 20 is a bar graph of the changes in HbA1c level at 90 days comparedto baseline for populations in groups 1 and 4 having a difference inHbA1c levels between screening and baseline of, for Group 1, 0-1.1,0-0.5 and 0-0.3 and, for Group 4, 0-1, 0-0.5 and 0-0.3.

FIG. 21 is a bar graph of the change in HbA1c values after 90 days(compared to baseline) for patients in groups 1 and 4 having particularbaseline HbA1c values and a difference in the HbA1c values fromscreening to baseline of 0-0.3. The first bar represents absolutechanges in HbA1c levels (t=0 vs. t=90) for those subjects in Group Ihaving baseline HbA1c levels ranging from 7 to 8.9 and a 0-0.3 variationof HbA1c levels between screening and at baseline. The second barrepresents absolute changes in HbA1c levels for a subpopulation of Group1 having baseline HbA1c levels of 7.5 to 8.9 and a 0-0.3 variation ofHbA1c levels between screening and at baseline. The third bar representsa still narrower subpopulation—those patients of Group I having baselineHbA1c levels of 8 to 8.9 and a 0-0.3 variation of HbA1c levels betweenscreening and at baseline.

FIGS. 22-23 compare the changes in HbA1c levels for Groups 1 and 4 forthose patients having a 0-0.3 variation of HbA1c levels betweenscreening and at t=−0, with baseline HbA1c levels between 8 and 8.9. Forthis population, FIG. 22 depicts changes in HbA1c levels across the 90days for groups 1 and 4 and FIG. 23 depicts the number of patientsreaching specified HbA1c target levels at the end of the study. FIG. 24sets forth changes from baseline for C-Peptide, FBG, Fructosamine,HbA1c, Insulin and Proinsulin.

Amongst the participants in the study there were no significant adverseevents, no episodes of severe hypoglycemia, or weight gain amongstgroups I-III. Incidents of mild to moderate hypoglycemia and antibodiesin groups 1-3 were comparable to those found in group 4 (placebo).

1-64. (canceled)
 65. A solid dosage form wherein a delivery agent islyophilized with insulin or an insulin analog.
 66. A solid dosage formwherein a delivery agent is rotary evaporated with insulin or an insulinanalog.
 67. (canceled)
 68. A solid dosage form wherein a delivery agentand insulin or an insulin analog is in the form of a gel prior to beingtableted or filled in a capsule.
 69. The solid dosage form of claim 68comprising a gelatin.
 70. The compositions of any one of claim 65wherein the delivery agent is 4-CNAB. 71-73. (canceled)
 74. A processfor preparing a solid dosage form of claim 65 comprising the steps of(a) preparing an insulin/delivery agent solution; (b) freeze-drying theinsulin/delivery agent solution; (c) milling the insulin/delivery agentcolyophilized powder obtained by freeze-drying the insulin/deliveryagent solution; (d) mixing the milled co-lyophilized powder withintragranular excipients; (e) performing dry granulation, (f) addingextragranular excipients; and (g) forming a unit dosage form from theresulting composition.
 75. A process for preparing a solid dosage formof claim 66 comprising the steps of (a) preparing an insulin/deliveryagent solution; (b) performing rotary evaporation on theinsulin/delivery agent solution; and (c) forming a unit dosage form fromthe rotary evaporated insulin/delivery agent solution.
 76. The processof claim 75 further comprising at least one of the steps of (c) millingthe insulin/delivery agent powder obtained by rotary evaporation of theinsulin/delivery agent solution; (d) mixing the milled powder withintragranular excipients; (e) granulating the milled powder andintragranular excipients; and (f) adding extragranular excipients.
 77. Aprocess for preparing a solid dosage form of claim 68 comprising thestep of mixing a gelling agent with (a) a delivery agent compound and(b) insulin or an insulin analog.
 78. The process of claim 77 whereinthe gelling agent is gelatin.
 79. The process of any one of claim 77wherein the delivery agent is 4-CNAB or a salt thereof.
 80. The processof claim 79 wherein the delivery agent is sodium 4-CNAB. 81-91.(canceled)